Treatments for Gastrointestinal Disorders

ABSTRACT

The present invention features peptides, compositions, and related methods for treating gastrointestinal disorders and conditions, including but not limited to, irritable bowel syndrome (IBS), gastrointestinal motility disorders, functional gastrointestinal disorders, gastroesophageal reflux disease (GERD), duodenogastric reflux, Crohn&#39;s disease, ulcerative colitis, inflammatory bowel disease, functional heartburn, dyspepsia, visceral pain, gastroparesis, chronic intestinal pseudo-obstruction (or colonic pseudo-obstruction), disorders and conditions associated with constipation, and other conditions and disorders are described herein. using peptides and other agents that activate the guanylate cyclase C (GC-C) receptor.

PRIORITY CLAIM

This application is a continuation of, and claims priority to U.S.patent application Ser. No. 18/055,536 filed Nov. 15, 2022, which is acontinuation of U.S. patent application Ser. No. 17/716,560 filed Apr.8, 2022, which is a continuation of U.S. patent application Ser. No.17/459,451 filed Aug. 27, 2021, which is a continuation of U.S. patentapplication Ser. No. 17/140,267 filed Jan. 4, 2021, which is acontinuation of U.S. patent application Ser. No. 16/881,198 filed May22, 2020, which is a continuation of U.S. patent application Ser. No.16/585,139 filed Sep. 27, 2019, which is a continuation of U.S. patentapplication Ser. No. 16/270,802 filed Feb. 8, 2019, which is acontinuation of U.S. patent application Ser. No. 16/009,560 filed Jun.15, 2018, which is a continuation of U.S. patent application Ser. No.15/729,875 filed Oct. 11, 2017, which is a continuation of U.S. patentapplication Ser. No. 14/556,573 filed Dec. 1, 2014, which is acontinuation of U.S. patent application Ser. No. 13/579,685 filed Aug.17, 2012, which is the United States National Phase application ofPCT/US2011/025274 filed Feb. 17, 2011. This application also claimspriority to U.S. Provisional Application Ser. No. 61/305,465 filed Feb.17, 2010. The entire contents of the aforementioned applications areincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to peptides, compositions and methods fortreating gastrointestinal disorders.

SEQUENCE LISTING

This application incorporates by reference in its entirety the SequenceListing entitled “IW087PCT1US1CON1_ST25.txt” which is 630 bytes in sizeand last modified on Dec. 1, 2014 and filed electronically herewith.

BACKGROUND

Gastrointestinal disorders (GI) include irritable bowel syndrome (IBS)which is a common chronic disorder of the intestine that affects 20 to60 million individuals in the US alone (Lehman Brothers, GlobalHealthcare-Irritable bowel syndrome industry update, September 1999).IBS is the most common disorder diagnosed by gastroenterologists andaccounts for 12% of visits to primary care physicians (Camilleri 2001,Gastroenterology 120:652-668). In the US, the economic impact of IBS isestimated at $25 billion annually, through direct costs of health careuse and indirect costs of absenteeism from work (Talley 1995,Gastroenterology 109:1736-1741). Patients with IBS have three times moreabsenteeism from work and report a reduced quality of life. There is atremendous unmet medical need for patients suffering for IBS since fewprescription options exist to treat IBS.

Patients with IBS suffer from abdominal pain and a disturbed bowelpattern. Three subgroups of IBS patients have been defined based on thepredominant bowel habit: constipation-predominant irritable bowelsyndrome (c-IBS), diarrhea-predominant irritable bowel syndrome (d-IBS)or alternating between the two irritable bowel syndromes (a-IBS).Estimates of individuals who suffer from c-IBS range from 20-50% of theIBS patients with 30% frequently cited. In contrast to the other twosubgroups that have a similar gender ratio, c-IBS is more common inwomen (ratio of 3:1) (Talley et al. 1995, Am J Epidemiol 142:76-83).

The definition and diagnostic criteria for IBS have been formalized inthe “Rome Criteria” (Drossman et al. 1999, Gut 45:Suppl II: 1-81), whichare well accepted in clinical practice. Recently, there has beenincreasing evidence for a role of inflammation in etiology of IBS.Reports indicate that subsets of IBS patients have small but significantincreases in colonic inflammatory and mast cells, increased induciblenitric oxide (NO) and synthase (iNOS) and altered expression ofinflammatory cytokines (reviewed by Talley 2000, Medscape Coverage ofDDW week).

Gastrointestinal disorders can also include constipation wherein as manyas 34 million Americans suffer from symptoms associated with chronicconstipation (CC) and 8.5 million patients have sought treatment.Patients with CC often experience hard and lumpy stools, strainingduring defecation, a sensation of incomplete evacuation, and fewer thanthree bowel movements per week. The discomfort and bloating of CCsignificantly affects patients' quality of life by impairing theirability to work and participate in typical daily activities.

Half of CC patients are not satisfied with currently availabletreatments for CC. Thus, there remains a need for new compounds andmethods of treating CC.

U.S. Pat. Nos. 7,304,036 and 7,371,727 disclose peptides that act asagonists of the guanylate cyclase C (GC-C) receptor for the treatment ofgastrointestinal disorders. One particular peptide disclosed islinaclotide, which consists of the following amino acid sequence: CysCys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr. These patents alsodisclose methods for preparing linaclotide and related peptides.

Linaclotide has the amino acid structure of:

Linaclotide is orally administered and currently in clinical trials fortreatment of irritable bowel syndrome with constipation (IBS-c) andchronic constipation (CC), has numerous effects on GI physiologyincluding: (1) reduced visceral pain, (2) reduced bloating, and (3)increased GI transit, which can lead to increased stool frequency andimproved stool consistency. Orally administered linaclotide acts locallyby activating GC-C at the luminal surface; there are no detectablelevels of linaclotide seen systemically after oral administration attherapeutic dose levels. Thus, the results from clinical trials oflinaclotide, as well as preclinical studies that have been done withlinaclotide and related peptides, suggest that GC-C peptide agonists maybe used therapeutically.

The contents of the U.S. Pat. Nos. 7,304,036 and 7,371,727 areincorporated herein by reference in their entirety.

The present invention feature peptides which may be modified at theiramine groups into imidazolidinone derivatives and/or modified at theircarboxyl groups into alkyl esters that are capable of activating and/orbinding the guanylate cyclase-C(GC-C) receptors at different affinities.GC-C is a key regulator in mammals of intestinal function, although lowlevels of GC-C have been detected in other tissues. GC-C responds to theendogenous hormones, guanylin and uroguanylin, and to enteric bacterialpeptides from the heat stable enterotoxin family (ST peptide). Whenagonists bind to GC-C, there is an elevation of the second messenger,cyclic GMP (c-GMP), and an increase in chloride and bicarbonatesecretion, resulting in an increase in intestinal fluid secretion. Insome examples of the present invention, the peptides described hereinmay produce increased elevation of c-GMP levels and provide atherapeutic option for treating gastrointestinal disorders.

SUMMARY

The present invention features peptides, compositions, and relatedmethods for treating gastrointestinal disorders and conditions,including but not limited to, irritable bowel syndrome (MS)gastrointestinal motility disorders, constipation, functionalgastrointestinal disorders, gastroesophageal reflux disease (GERD),duodenogastric reflux, Crohn's disease, ulcerative colitis, inflammatorybowel disease, functional heartburn, dyspepsia, visceral pain,gastroparesis, chronic intestinal pseudo-obstruction (or colonicpseudo-obstruction), and other conditions and disorders described hereinusing peptides and compositions that activate the guanylate cyclase C(GC-C) receptor.

One aspect of the present invention provides a peptide or apharmaceutically acceptable salt thereof, wherein the peptide comprisesthe amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr, wherein at least one carboxyl group of the peptide is an alkylester having the formula (—COOR) in which R is a C₁₋₆ alkyl.

In one embodiment, the peptide comprises the amino acid structure of:

or a pharmaceutically acceptable salt thereof, wherein R′ is H or a C₁₋₆alkyl, and at least one R′ is C₁₋₆ alkyl.

A second aspect of the present invention provides a peptide thatcomprises the amino acid structure of:

or a pharmaceutically acceptable salt thereof, wherein R is a C₁₋₆alkyl; or

a peptide that comprises the amino acid structure of:

or a pharmaceutically acceptable salt thereof, wherein R is C₁₋₆ alkyl.

A third aspect of the present invention provides a peptide orpharmaceutically acceptable salt thereof, wherein the peptide comprisesthe amino acid structure of:

A fourth aspect of the present invention provides a pharmaceuticalcomposition comprising a peptide of the present invention.

A fifth aspect of the present invention provides a method for treating agastrointestinal disorder, which includes administering a pharmaceuticalcomposition according to the present invention.

The details of one or more embodiments of the invention are set forth inthe accompanying description.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 shows the dose response of exemplary peptides of the presentinvention in a T84 cell c-GMP assay.

FIG. 2 demonstrates an example of an analysis of exemplary peptides byRP-HPLC, wherein “Cys₁-IMD” refers to the linaclotide imidazolidinonederivative modified on its N-terminal amine group.

FIG. 3 shows specific binding of linaclotide and Cys₁-IMD tocell-surface GC-C receptors on T84 cells in a competitive radioligandbinding assay.

FIG. 4 shows the dose response of exemplary peptides of the presentinvention in a T84 cell c-GMP assay.

The figures are provided by way of example and are not intended to limitthe scope of the present invention.

DETAILED DESCRIPTION

Guanylate cyclase C (GC-C) is a transmembrane receptor that is locatedon the apical surface of epithelial cells in the stomach and intestine.The receptor has an extracellular ligand-binding domain, a singletransmembrane region and a C-terminal guanylyl cyclase domain. When aligand binds to the extracellular domain of GC-C, the intracellularcatalytic domain catalyzes the production of cGMP from GTP. In vivo,this increase in intracellular cGMP initiates a cascade of events thatleads to increased secretion of chloride and bicarbonate into theintestinal lumen, increased luminal pH, decreased luminal sodiumabsorption, increased fluid secretion, and acceleration of intestinaltransit. cGMP is secreted bidirectionally from the epithelium into themucosa and lumen. The peptides and compositions of the present inventionbind to the intestinal GC-C receptor which is a regulator of fluid andelectrolyte balance in the intestine.

In some circumstances it can be desirable to treat patients with avariant or modified peptide that binds to and activates intestinal GC-Creceptors, but is less active or more active than the non-variant formof a peptide. Reduced activity can arise from reduced affinity for thereceptor or a reduced ability to activate the receptor once bound orreduced stability of the peptide. Increased activity can arise fromincreased affinity for the receptor or an increased ability to activatethe receptor once bound or increased stability of the peptide.

Description of Exemplary Peptides:

In various embodiments, a peptide may be modified wherein at least onecarboxyl group of the amino acid residue of the peptide is modified toan alkyl ester. This modification may be produced, for example, bytreating a carboxylic acid with an alcohol in the presence of adehydrating agent wherein the dehydrating agent can include but is notlimited to a strong acid such as sulfuric acid. Other methods ofproducing alkyl esters from carboxyl groups are readily known in thoseskilled in the arts and are incorporated herein.

As used herein, a carboxyl group has the formula: (—COOH).

As used herein, the term “alkyl”, refers to a saturated linear orbranched-chain monovalent hydrocarbon radical.

As used herein, a group is terminal or terminus when the group ispresent at the end of the amino acid sequence.

As used herein, an amine group on a peptide has the formula:

wherein R² is the rest of the peptide.

As used herein, an imine group on a peptide has the formula:

wherein R² is the rest of the peptide.

In some embodiments, the carboxylic acid of the side chain of aglutamate amino acid in a peptide sequence is modified into an alkylester.

In further embodiments, the carboxylic acid on the side chain of aglutamate amino acid a peptide sequence is modified into an ethyl ester.

In other embodiments, the C-terminus carboxylic acid of a tyrosine aminoacid in a peptide sequence is modified into an alkyl ester.

In further embodiments, the C-terminus carboxylic acid of a tyrosineamino acid of a peptide sequence is modified into an ethyl ester.

In several embodiments, the present invention provides a peptide or apharmaceutically acceptable salt thereof, wherein the peptide comprisesthe amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr, wherein at least one carboxyl group of the peptide is an alkylester having the formula (—COOR) in which R is a C₁₋₆ alkyl.

In several embodiments, the peptide comprises an amino acid structureof:

or a pharmaceutically acceptable salt thereof, wherein R′ is H or a C₁₋₆alkyl, and at least one R′ is C₁₋₆ alkyl.

In some embodiments, the peptide or pharmaceutically acceptable saltthereof comprises a peptide having an amino acid structure of:

wherein R is a C₁₋₆ alkyl (“Glu₃-alkyl ester”).

In other embodiments, R is a C₁₋₄ alkyl.

In further embodiments, R is methyl, ethyl, or propyl.

In some embodiments, the peptide or pharmaceutically acceptable saltthereof comprises an amino acid structure of (“Glu₃-ethyl ester”):

In some embodiments, the peptide or pharmaceutically acceptable saltthereof comprises an amino acid structure of:

In some embodiments, the peptide or pharmaceutically acceptable saltthereof comprises an amino acid structure of:

In some embodiments, the C-terminal tyrosine of the Glu₃-alkyl ester orpharmaceutically acceptable salt is absent.

In some embodiments, the peptide or pharmaceutically acceptable saltthereof comprises an amino acid structure of:

wherein R is C₁₋₆ alkyl.

In other embodiments, R is a C₁₋₄ alkyl.

In further embodiments, R is methyl, ethyl, or propyl.

In some embodiments, the peptide or pharmaceutically acceptable saltthereof comprises an amino acid structure of (“Tyr₁₄-ethyl ester”):

In some embodiments, the peptide or pharmaceutically acceptable saltthereof comprises an amino acid structure of:

In some embodiments, the peptide or pharmaceutically acceptable saltthereof comprises an amino acid structure of:

Without wishing to be bound by any theory, a peptide may be modifiedwherein at least one amine group of the amino acid residues of thepeptide is modified into an imine. This modification may be produced,for example, by treating an amine group with a carbonyl, such as analdehyde or ketone, in the presence of acid catalyst. Other methods ofproducing imines from amine groups are readily known to those skilled inthe arts and are incorporated herein.

In some embodiments, the imine modification may be produced by aformaldehyde mediated reaction in the presence of acid catalyst.

In further embodiments, the imine carbon may be cross-linked to anotheramine group of the peptide.

In other embodiments, a peptide may be modified into an imine at theα-amine group of the N-terminal amino acid, wherein the imine carbon iscross-linked with an amine group of the second amino acid residue of thepeptide forming a five membered ring.

In other embodiments, a peptide comprising the amino acid sequence CysCys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr may be modified withthe addition of methylene at the α-amine group of the N-terminal Cys₁which is cross-linked to the amine group of Cyst to form animidazolidinone 5 membered ring at the N-terminus of the peptide(“Cys₁-IMD”).

In one aspect, the invention provides novel GC-C peptide agonists usefulfor the treatment of gastrointestinal disorders.

In several embodiments, the present invention provides a peptide or apharmaceutically acceptable salt thereof, wherein the peptide comprisesthe amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys Tyr, wherein at least one amine group of the peptide is an iminehaving the formula

wherein R² is the rest of the peptide.

In some embodiments, the peptide or a pharmaceutically acceptable saltcomprises a peptide wherein the N-terminal amine group of the peptide isan imine having the formula

wherein R² is the rest of the peptide.

In further embodiments, the peptide or pharmaceutically acceptable saltthereof comprises an amino acid structure of:

In several embodiments, the peptide or pharmaceutically acceptable saltthereof comprises an amino acid structure of:

In some embodiments, the C-terminal tyrosine of the Cys₁-IMD peptide orpharmaceutically acceptable salt thereof is absent. In some embodiments,the Cys₁-IMD peptide or pharmaceutically acceptable salt thereof furthercomprises one or more peptide modifications, wherein at least onecarboxyl group of the peptide is an alkyl ester having the formula(—COOR) in which R is a C₁₋₆ alkyl.

In several embodiments, the present invention provides a peptide or apharmaceutically acceptable salt thereof, wherein the peptide consistsof the amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys ThrGly Cys Tyr, wherein at least one carboxyl group of the peptide is analkyl ester having the formula (—COOR) in which R is a C₁₋₆ alkyl.

In several embodiments, the peptide consists of an amino acid structureof:

or a pharmaceutically acceptable salt thereof, wherein R′ is H or a C₁₋₆alkyl, and at least one R′ is C₁₋₆ alkyl.

In some embodiments, the peptide or pharmaceutically acceptable saltthereof consists of an amino acid structure of:

wherein R is a C₁₋₆ alkyl. In further embodiments, the C-terminaltyrosine is absent.

In other embodiments, R is a C₁₋₄ alkyl.

In further embodiments, R is methyl, ethyl, or propyl.

In some embodiments, the peptide or pharmaceutically acceptable saltthereof consists of a peptide having an amino acid structure of:

In some embodiments, the peptide or pharmaceutically acceptable saltthereof consists of a peptide having an amino acid structure of:

In some embodiments, the peptide or pharmaceutically acceptable saltthereof consists of a peptide having an amino acid structure of:

In some embodiments, the peptide or pharmaceutically acceptable saltthereof consists of a peptide having an amino acid structure of:

wherein R is C₁₋₆ alkyl.

In other embodiments, R is a C₁₋₄ alkyl.

In further embodiments, R is methyl, ethyl, or propyl.

In some embodiments, the peptide or pharmaceutically acceptable saltthereof consists of an amino acid structure of:

In some embodiments, the peptide or pharmaceutically acceptable saltthereof consists of an amino acid structure of:

In some embodiments, the peptide or pharmaceutically acceptable saltthereof consists of an amino acid structure of:

In several embodiments, the present invention provides a peptide or apharmaceutically acceptable salt thereof, wherein the peptide consistsof the amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys ThrGly Cys Tyr, wherein at least one amine group of the peptide is an iminehaving the formula

wherein R² is the rest of the peptide.

In some embodiments, the peptide or a pharmaceutically acceptable saltconsists of a peptide wherein the N-terminal amine group of the peptideis an imine having the formula

wherein R² is the rest of the peptide.

In further embodiments, the peptide or pharmaceutically acceptable saltthereof consists of an amino acid structure of:

In some embodiments, the peptide or pharmaceutically acceptable saltthereof consists of an amino acid structure of:

In some embodiments, the C-terminal tyrosine of the Cys₁-IMD peptide orpharmaceutically acceptable salt thereof is absent. In some embodiments,the Cys₁-IMD peptide or pharmaceutically acceptable salt thereof furthercomprises one or more peptide modifications, e.g., wherein at least onecarboxyl group of the peptide is an alkyl ester having the formula(—COOR), wherein R is a C₁₋₆ alkyl.

In some embodiments, the peptide or pharmaceutically acceptable saltthereof activates the guanylate cyclase C receptor.

In other embodiments, the peptide or pharmaceutically acceptable saltthereof comprises 30 or fewer amino acids.

In further embodiments, the peptide or pharmaceutically acceptable saltthereof comprises 20 or fewer amino acids.

In other embodiments, the peptide or pharmaceutically acceptable saltthereof comprises a peptide wherein fewer than five amino acids precedethe first Cys residue of the amino acid sequence.

In some embodiments, the peptide or pharmaceutically acceptable saltthereof is isolated.

In other embodiments, the peptide or pharmaceutically acceptable saltthereof is purified.

In some embodiments, a pharmaceutically acceptable salt of the peptideis provided. In some instances, the pharmaceutically acceptable salt isa chloride salt.

Variant or Modified Peptides

In various embodiments, the peptide includes two Cys that form onedisulfide bond, the peptide includes four Cys that form two disulfidebonds, or the peptide includes six Cys that form three disulfide bonds.

In some peptides one or both members of one or both pairs of Cysresidues which normally form a disulfide bond can be replaced byhomocysteine, penicillamine, 3-mercaptoproline (Kolodziej et al. 1996Int J Pept Protein Res 48:274); β, β dimethylcysteine (Hunt et al. 1993Int J Pept Protein Res 42:249) or diaminopropionic acid (Smith et al.1978 J Med Chem 21:117) to form alternative internal cross-links at thepositions of the normal disulfide bonds. In other embodiments, thedisulfide bonds may be replaced by hydrocarbon crosslinking(Schafmeister et al. 2000 J Am Chem Soc 122:5891, Patgiri et al. 2008Acc Chem Res 41:1289, Henchey et al. 2008 Curr Opin Chem Biol 12:692).

Production of Peptides

In one embodiment, peptides or precursor peptides of the invention canbe produced recombinantly in any known protein expression system,including, without limitation, bacteria (e.g., E. coli or Bacillussubtilis), insect cell systems (e.g., Drosophila Sf9 cell systems),yeast cell systems (e.g., S. cerevisiae, S. saccharomyces) orfilamentous fungal expression systems, or animal cell expression systems(e.g., mammalian cell expression systems). Peptides or precursorpeptides of the invention may also be chemically synthesized.

If the peptide or variant peptide is to be produced recombinantly, e.g.,E. coli, the nucleic acid molecule encoding the peptide may also encodea leader sequence that permits the secretion of the mature peptide fromthe cell. Thus, the sequence encoding the peptide can include the presequence and the pro sequence of, for example, a naturally-occurringbacterial ST peptide. The secreted, mature peptide can be purified fromthe culture medium.

The sequence encoding a peptide described herein can be inserted into avector capable of delivering and maintaining the nucleic acid moleculein a bacterial cell. The DNA molecule may be inserted into anautonomously replicating vector (suitable vectors include, for example,pGEM3Z and pcDNA3, and derivatives thereof). The vector nucleic acid maybe a bacterial or bacteriophage DNA such as bacteriophage lambda or M13and derivatives thereof. Construction of a vector containing a nucleicacid described herein can be followed by transformation of a host cellsuch as a bacterium. Suitable bacterial hosts include but are notlimited to, E. coli, B. subtilis, Pseudomonas and Salmonella. Thegenetic construct also includes, in addition to the encoding nucleicacid molecule, elements that allow expression, such as a promoter andregulatory sequences. The expression vectors may contain transcriptionalcontrol sequences that control transcriptional initiation, such aspromoter, enhancer, operator, and repressor sequences. A variety oftranscriptional control sequences are well known to those in the art.The expression vector can also include a translation regulatory sequence(e.g., an untranslated 5′ sequence, an untranslated 3′ sequence, or aninternal ribosome entry site). The vector can be capable of autonomousreplication or it can integrate into host DNA to ensure stability duringpeptide production.

The protein coding sequence that includes a peptide described herein canalso be fused to a nucleic acid encoding a peptide affinity tag, e.g.,glutathione S-transferase (GST), maltose E binding protein, protein A,FLAG tag, hexa-histidine, myc tag or the influenza HA tag, in order tofacilitate purification. The affinity tag or reporter fusion joins thereading frame of the peptide of interest to the reading frame of thegene encoding the affinity tag such that a translational fusion isgenerated. Expression of the fusion gene results in translation of asingle peptide that includes both the peptide of interest and theaffinity tag. In some instances where affinity tags are utilized, DNAsequence encoding a protease recognition site will be fused between thereading frames for the affinity tag and the peptide of interest.

Genetic constructs and methods suitable for production of immature andmature forms of the peptides and variants described herein in proteinexpression systems other than bacteria, and well known to those skilledin the art, can also be used to produce peptides in a biological system.

In other embodiments, peptides containing amino acids not normallyincorporated by the translation machinery and described above(e.g.—β-carboxylated Asp, γ-carboxylated Glu, Asu, Aad and Apm) may berecombinantly produced by tRNA modification methods. Methods formodifying tRNA including, but not limited to, modifying the anti-codon,the amino acid attachment site, and/or the accepter stem to allowincorporation of unnatural and/or arbitrary amino acids are known in theart (Biochem. Biophys. Res. Comm. (2008) 372: 480-485; Chem. Biol.(2009) 16:323-36; Nat. Methods (2007) 4:239-44; Nat. Rev. Mol. CellBiol. (2006) 7:775-82; Methods (2005) 36:227-238; Methods (2005)36:270-278; Annu. Rev. Biochem. (2004) 73:147-176; Nuc. Acids Res.(2004) 32:6200-6211; Proc. Natl. Acad. Sci. USA (2003) 100:6353-6357;Royal Soc. Chem. (2004) 33:422-430).

In some embodiments, peptides may be chemically produced. Peptides canbe synthesized by a number of different methods including solution andsolid phase synthesis using traditional BOC or FMOC protection. Forexample, the peptide can be synthesized on 2-Chlorotrityl or Wang resinusing consecutive amino acid couplings. The following protecting groupscan be used: Fluorenylmethyloxycarbonyl or tert-butyloxycarbonyl(alpha-amino groups, N-terminus); trityl or tert-butyl (thiol groups ofCys); tert-butyl (γ-carboxyl of glutamic acid and the hydroxyl group ofthreonine, if present); and trityl (β-amid function of the asparagineside chain and the phenolic group of tyrosine, if present). Coupling canbe effected with DIC and HOBt in the presence of a tertiary amine, andthe peptide can be deprotected and cleaved from the solid support inusing cocktail K (trifluoroacetic acid 81%, phenol 5%, thioanisole 5%,1,2-ethanedithiol 2.5%, water 3%, dimethylsulphide 2%, ammonium iodide1.5% w/w). After removal of trifluoroacetic acid and other volatiles thepeptide can be precipitated using an organic solvent. Disulfide bondsbetween Cys residues can be formed using dimethyl sulfoxide (Tam et al.(1991) J. Am. Chem. Soc. 113:6657-62) or using an air oxidationstrategy. The resulting peptide can be purified by reverse-phasechromatography and lyophilized.

These peptides can be made, isolated or used either in form of the baseor as pharmaceutically acceptable salts thereof. Examples of saltsinclude, without limitation, acetate, chloride, sulfate and phosphatesalts of the peptide.

Compositions of Peptides and GC-C Receptor Agonists

In another aspect, pharmaceutical compositions are provided wherein thepeptides, alone or in combination, can be combined with anypharmaceutically acceptable carrier or medium.

In several embodiments, the pharmaceutical composition comprises apeptide or pharmaceutically acceptable salt thereof as described herein.The pharmaceutical composition may comprise two or more peptides orpharmaceutically acceptable salts thereof described herein.

In some embodiments, the pharmaceutical composition comprises two ormore peptides selected from:

-   -   i. a peptide or a pharmaceutically acceptable salt thereof,        wherein the peptide comprises the amino acid structure of:

-   -   ii. a peptide or a pharmaceutically acceptable salt thereof,        wherein the peptide comprises an amino acid structure of

and

-   -   iii. a peptide or a pharmaceutically acceptable salt thereof,        wherein the peptide comprises the amino acid sequence Cys Cys        Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr, wherein at        least one carboxyl group of the peptide is an alkyl ester having        the formula (—COOR) in which R is a C₁₋₆ alkyl.

In other embodiments, the pharmaceutical composition compriseslinaclotide and a peptide or a pharmaceutically acceptable salt thereof,wherein the peptide comprises an amino acid structure of:

and the peptide or pharmaceutically acceptable salt thereof comprisesless than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weightcompared to the weight of linaclotide.

In further embodiments, the pharmaceutical composition compriseslinaclotide and a peptide or a pharmaceutically acceptable salt thereof,wherein the peptide comprises an amino acid structure of:

and the peptide or pharmaceutically acceptable salt thereof comprisesless than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% by weight compared tothe weight of linaclotide.

In some embodiments, the imidazolidinone derivative of linaclotidecomprises less than about 15% by weight of the composition, less thanabout 10% by weight of the composition, less than about 7% by weight ofthe composition or less than about 5% by weight of the composition. Inother exemplary embodiments, the imidazolidinone derivative oflinaclotide comprises from about 0.01% to about 15% by weight of thecomposition, about 0.05% to about 10% by weight of the composition,about 0.05% to about 7% by weight of the composition or about 0.05% toabout 5% by weight of the composition.

In other embodiments, the pharmaceutical composition compriseslinaclotide and a peptide or a pharmaceutically acceptable salt thereof,wherein the peptide comprises an amino acid structure of:

and the peptide or pharmaceutically acceptable salt thereof comprisesless than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weightcompared to the weight of linaclotide.

In further embodiments, the pharmaceutical composition compriseslinaclotide and a peptide or a pharmaceutically acceptable salt thereof,wherein the peptide comprises an amino acid structure of:

and the peptide or pharmaceutically acceptable salt thereof comprisesless than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% by weight compared tothe weight of linaclotide.

In some embodiments, the Tyr₁₄-ethyl ester peptide comprises less thanabout 15% by weight of the composition, less than about 10% by weight ofthe composition, less than about 7% by weight of the composition or lessthan about 5% by weight of the composition. In other exemplaryembodiments, the Tyr₁₄-ethyl ester comprises from about 0.01% to about15% by weight of the composition, about 0.05% to about 10% by weight ofthe composition, about 0.05% to about 7% by weight of the composition orabout 0.05% to about 5% by weight of the composition.

In other embodiments, the pharmaceutical composition comprisinglinaclotide and a peptide or a pharmaceutically acceptable salt thereof,wherein the peptide comprises an amino acid structure of:

and the peptide or pharmaceutically acceptable salt thereof comprisesless than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weightcompared to the weight of linaclotide.

In further embodiments, the pharmaceutical composition compriseslinaclotide and a peptide or a pharmaceutically acceptable salt thereof,wherein the peptide comprises an amino acid structure of:

and the peptide or pharmaceutically acceptable salt thereof comprisesless than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% by weight compared tothe weight of linaclotide.

In some embodiments, the Glu₃-ethyl ester peptide comprises less thanabout 15% by weight of the composition, less than about 10% by weight ofthe composition, less than about 7% by weight of the composition or lessthan about 5% by weight of the composition. In other exemplaryembodiments, the Glu₃-ethyl ester comprises from about 0.01% to about15% by weight of the composition, about 0.05% to about 10% by weight ofthe composition, about 0.05% to about 7% by weight of the composition orabout 0.05% to about 5% by weight of the composition.

In some embodiments, the pharmaceutical composition comprises a peptideor pharmaceutically acceptable salt, wherein the peptide consists of theamino acid structure of:

In other embodiments, the pharmaceutical composition consistsessentially of a peptide or pharmaceutically acceptable salt thereof,wherein the peptide comprises the amino acid structure of:

The term “consisting essentially of”, and variants thereof, when used torefer to the composition, are used herein to mean that the compositionincludes a sole active peptide and other desired pharmaceuticallyinactive additives, excipients, and/or components (e.g., polymers,sterically hindered primary amines, cations, filling agents, binders,carriers, excipients, diluents, disintegrating additives, lubricants,solvents, dispersants, coating additives, absorption promotingadditives, controlled release additives, anti-caking additives,anti-microbial additives, preservatives, sweetening additives,colorants, flavors, desiccants, plasticizers, dyes, or the like), and noother active pharmaceutical ingredient(s).

The peptides described herein can be combined with any pharmaceuticallytolerable carrier or medium, e.g. solvents, dispersants, coatings,absorption promoting agents, controlled release agents, and one or moreinert excipients (which include starches, polyols, granulating agents,microcrystalline cellulose (e.g. celphere, Celphere beads®), diluents,lubricants, binders, disintegrating agents, and the like), etc. Ifdesired, tablet dosages of the disclosed compositions may be coated bystandard aqueous or nonaqueous techniques.

Examples of excipients for use as the pharmaceutically acceptablecarriers and the pharmaceutically acceptable inert carriers and theaforementioned additional ingredients include, but are not limited tobinders, fillers, disintegrants, lubricants, anti-microbial agents, andcoating agents.

As used herein, the term “binder” refers to any pharmaceuticallyacceptable binder that may be used in the practice of the invention.Examples of pharmaceutically acceptable binders include, withoutlimitation, a starch (e.g., corn starch, potato starch andpre-gelatinized starch (e.g., STARCH 1500® and STARCH 1500 LM®, sold byColorcon, Ltd.) and other starches), maltodextrin, gelatin, natural andsynthetic gums such as acacia, powdered tragacanth, guar gum, celluloseand its derivatives (e.g., methylcellulose, hydroxyethyl cellulose,hydroxyethyl methylcellulose, hydroxypropyl cellulose and hydroxypropylmethylcellulose (hypromellose), ethyl cellulose, cellulose acetate,carboxymethyl cellulose calcium, sodium carboxymethyl cellulose,carboxymethylcellulose, microcrystalline cellulose (e.g. AVICEL™ suchas, AVICEL-PH-101™, -103™ and 105™, sold by FMC Corporation, MarcusHook, PA, USA)), polyvinyl alcohol, polyvinyl pyrrolidone (e.g.,polyvinyl pyrrolidone K30), and mixtures thereof.

As used herein, the term “filler” refers to any pharmaceuticallyacceptable filler that may be used in the practice of the invention.Examples of pharmaceutically acceptable fillers include, withoutlimitation, talc, calcium carbonate (e.g., granules or powder), dibasiccalcium phosphate, tribasic calcium phosphate, calcium sulfate (e.g.,granules or powder), microcrystalline cellulose (e.g., Avicel PH101 orCelphere CP-305), powdered cellulose, dextrates, kaolin, mannitol,silicic acid, sorbitol, starch (e.g., Starch 1500), pre-gelatinizedstarch, lactose, glucose, fructose, galactose, trehalose, sucrose,maltose, isomalt, raffinose, maltitol, melezitose, stachyose, lactitol,palatinite, xylitol, myoinositol, and mixtures thereof.

Examples of pharmaceutically acceptable fillers that may be particularlyused for coating the peptides include, without limitation, talc,microcrystalline cellulose (e.g., Avicel PH101 or Celphere CP-305),powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol,starch, pre-gelatinized starch, lactose, glucose, fructose, galactose,trehalose, sucrose, maltose, isomalt, dibasic calcium phosphate,raffinose, maltitol, melezitose, stachyose, lactitol, palatinite,xylitol, mannitol, myoinositol, and mixtures thereof.

As used herein, the term “additives” refers to any pharmaceuticallyacceptable additive. Pharmaceutically acceptable additives include,without limitation, disintegrants, dispersing additives, lubricants,glidants, antioxidants, coating additives, diluents, surfactants,flavoring additives, humectants, absorption promoting additives,controlled release additives, anti-caking additives, anti-microbialagents (e.g., preservatives), colorants, desiccants, plasticizers anddyes. As used herein, an “excipient” is any pharmaceutically acceptableadditive, filler, binder or agent.

Compositions of the present invention may also optionally include othertherapeutic ingredients, anti-caking agents, preservatives, sweeteningagents, colorants, flavors, desiccants, plasticizers, dyes, glidants,anti-adherents, anti-static agents, surfactants (wetting agents),antioxidants, film-coating agents, and the like. Any such optionalingredient must be compatible with the compound described herein toinsure the stability of the formulation. The composition may containother additives as needed, including for example lactose, glucose,fructose, galactose, trehalose, sucrose, maltose, raffinose, maltitol,melezitose, stachyose, lactitol, palatinite, starch, xylitol, mannitol,myoinositol, and the like, and hydrates thereof, and amino acids, forexample alanine, glycine and betaine, and peptides and proteins, forexample albumen.

The compositions can include, for example, various additional solvents,dispersants, coatings, absorption promoting additives, controlledrelease additives, and one or more inert additives (which include, forexample, starches, polyols, granulating additives, microcrystallinecellulose, diluents, lubricants, binders, disintegrating additives, andthe like), etc. If desired, tablet dosages of the disclosed compositionsmay be coated by standard aqueous or non-aqueous techniques.Compositions can also include, for example, anti-caking additives,preservatives, sweetening additives, colorants, flavors, desiccants,plasticizers, dyes, and the like.

Suitable disintegrants include, for example, agar-agar, calciumcarbonate, microcrystalline cellulose, croscarmellose sodium,crospovidone, povidone, polacrilin potassium, sodium starch glycolate,potato or tapioca starch, other starches, pre-gelatinized starch, clays,other algins, other celluloses, gums, and mixtures thereof.

Suitable lubricants include, for example, calcium stearate, magnesiumstearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol,polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate,talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil,sunflower oil, sesame oil, olive oil, corn oil and soybean oil), zincstearate, ethyl oleate, ethyl laurate, agar, syloid silica gel (AEROSIL200, W.R. Grace Co., Baltimore, MD USA), a coagulated aerosol ofsynthetic silica (Evonik Degussa Co., Plano, TX USA), a pyrogenicsilicon dioxide (CAB-O-SIL, Cabot Co., Boston, MA USA), and mixturesthereof.

Suitable glidants include, for example, leucine, colloidal silicondioxide, magnesium trisilicate, powdered cellulose, starch, talc, andtribasic calcium phosphate.

Suitable anti-caking additives include, for example, calcium silicate,magnesium silicate, silicon dioxide, colloidal silicon dioxide, talc,and mixtures thereof.

Suitable anti-microbial additives that may be used, e.g., as apreservative for the peptides compositions, include, for example,benzalkonium chloride, benzethonium chloride, benzoic acid, benzylalcohol, butyl paraben, cetylpyridinium chloride, cresol, chlorobutanol,dehydroacetic acid, ethylparaben, methylparaben, phenol, phenylethylalcohol, phenoxyethanol, phenylmercuric acetate, phenylmercuric nitrate,potassium sorbate, propylparaben, sodium benzoate, sodiumdehydroacetate, sodium propionate, sorbic acid, thimersol, thymo, andmixtures thereof.

Suitable antioxidants include, for example, BHA (butylatedhydroxyanisole), BHT (butylated hydroxytoluene), vitamin E, propylgallate, ascorbic acid and salts or esters thereof, tocopherol andesters thereof, alpha-lipoic acid and beta-carotene.

Suitable coating additives include, for example, sodium carboxymethylcellulose, cellulose acetate phthalate, ethylcellulose, gelatin,pharmaceutical glaze, hydroxypropyl cellulose, hydroxypropylmethylcellulose, hydroxypropyl methyl cellulose phthalate,methylcellulose, polyethylene glycol, polyvinyl acetate phthalate,shellac, sucrose, titanium dioxide, carnauba wax, microcrystalline wax,and mixtures thereof. Suitable protective coatings include Aquacoat(e.g. Aquacoat Ethylcellulose Aquaeous Dispersion, 15% w/w, FMCBiopolymer, ECD-30), Eudragit (e.g. Eudragit E PO PE-EL, Roehm PharmaPolymers) and Opadry (e.g Opadry AMB dispersion, 20% w/w, Colorcon).

In certain embodiments, suitable additives for the peptides compositioninclude one or more of sucrose, talc, magnesium stearate, crospovidoneor BHA.

The compositions of the present invention can also include otherexcipients, agents, and categories thereof including but not limited toL-histidine, Pluronic®, Poloxamers (such as Lutrol® and Poloxamer 188),ascorbic acid, glutathione, permeability enhancers (e.g. lipids, sodiumcholate, acylcarnitine, salicylates, mixed bile salts, fatty acidmicelles, chelators, fatty acid, surfactants, medium chain glycerides),protease inhibitors (e.g. soybean trypsin inhibitor, organic acids), pHlowering agents and absorption enhancers effective to promotebioavailability (including but not limited to those described in U.S.Pat. Nos. 6,086,918 and 5,912,014), materials for chewable tablets (likedextrose, fructose, lactose monohydrate, lactose and aspartame, lactoseand cellulose, maltodextrin, maltose, mannitol, microcrystallinecellulose and guar gum, sorbitol crystalline); parenterals (likemannitol and povidone); plasticizers (like dibutyl sebacate,plasticizers for coatings, polyvinylacetate phthalate); powderlubricants (like glyceryl behenate); soft gelatin capsules (likesorbitol special solution); spheres for coating (like sugar spheres);spheronization agents (like glyceryl behenate and microcrystallinecellulose); suspending/gelling agents (like carrageenan, gellan gum,mannitol, microcrystalline cellulose, povidone, sodium starch glycolate,xanthan gum); sweeteners (like aspartame, aspartame and lactose,dextrose, fructose, honey, maltodextrin, maltose, mannitol, molasses,sorbitol crystalline, sorbitol special solution, sucrose); wetgranulation agents (like calcium carbonate, lactose anhydrous, lactosemonohydrate, maltodextrin, mannitol, microcrystalline cellulose,povidone, starch), caramel, carboxymethylcellulose sodium, cherry creamflavor and cherry flavor, citric acid anhydrous, citric acid,confectioner's sugar, D&C Red No. 33, D&C Yellow #10 Aluminum Lake,disodium edetate, ethyl alcohol 15%, FD& C Yellow No. 6 aluminum lake,FD&C Blue #1 Aluminum Lake, FD&C Blue No. 1, FD&C blue no. 2 aluminumlake, FD&C Green No. 3, FD&C Red No. 40, FD&C Yellow No. 6 AluminumLake, FD&C Yellow No. 6, FD&C Yellow No. 10, glycerol palmitostearate,glyceryl monostearate, indigo carmine, lecithin, manitol, methyl andpropyl parabens, mono ammonium glycyrrhizinate, natural and artificialorange flavor, pharmaceutical glaze, poloxamer 188, Polydextrose,polysorbate 20, polysorbate 80, polyvidone, pregelatinized corn starch,pregelatinized starch, red iron oxide, saccharin sodium, sodiumcarboxymethyl ether, sodium chloride, sodium citrate, sodium phosphate,strawberry flavor, synthetic black iron oxide, synthetic red iron oxide,titanium dioxide, and white wax.

In some embodiments, there is provided a pharmaceutical compositioncomprising a peptide described herein and one or more agents selectedfrom Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺, K⁺, Na⁺ or Al³⁺, a combination thereof,and/or a sterically hindered primary amine. In further embodiments, theagent is Mg²⁺, Ca²⁺ or Zn²⁺ or a combination thereof. In someembodiments, the cation is provided, without limitation, as magnesiumacetate, magnesium chloride, magnesium phosphate, magnesium sulfate,calcium acetate, calcium chloride, calcium phosphate, calcium sulfate,zinc acetate, zinc chloride, zinc phosphate, zinc sulfate, manganeseacetate, manganese chloride, manganese phosphate, manganese sulfate,potassium acetate, potassium chloride, potassium phosphate, potassiumsulfate, sodium acetate, sodium chloride, sodium phosphate, sodiumsulfate, aluminum acetate, aluminum chloride, aluminum phosphate oraluminum sulfate. In further embodiments, the cation is provided asmagnesium chloride, calcium chloride, calcium phosphate, calciumsulfate, zinc acetate, manganese chloride, potassium chloride, sodiumchloride or aluminum chloride. In other embodiments, the cation isprovided as calcium chloride, magnesium chloride or zinc acetate.

In another embodiment, the agent is a sterically hindered primary amine.In a further embodiment, the sterically hindered primary amine is anamino acid. In yet a further embodiment, the amino acid is anaturally-occurring amino acid. In a still further embodiment, thenaturally-occurring amino acid is selected from the group consisting of:histidine, phenylalanine, alanine, glutamic acid, aspartic acid,glutamine, leucine, methionine, asparagine, tyrosine, threonine,isoleucine, tryptophan, glycine and valine; yet further, thenaturally-occurring amino acid is leucine, isoleucine, alanine ormethionine. In a still further embodiment, the naturally-occurring aminoacid is leucine. In another embodiment, the sterically hindered primaryamine is a non-naturally occurring amino acid (e.g., 1-aminocyclohexanecarboxylic acid). In a further embodiment, the sterically hinderedprimary amine is cyclohexylamine, 2-methylbutylamine or chitosan. Inanother embodiment, one or more sterically hindered primary amines maybe used in a composition.

In some cases, the sterically hindered primary amine has the formula:

wherein R₁, R₂ and R₃ are independently selected from: H, C(O)OH, C1-C6alkyl, C1-C6 alkylether, C1-C6 alkylthioether, C1-C6 alkyl carboxylicacid, C1-C6 alkyl carboxylamide and alkylaryl, wherein any group can besingly or multiply substituted with: halogen or amino, and provided thatno more than two of R₁, R₂ and R₃ are H. In another embodiment, no morethan one of R₁, R₂ and R₃ is H.

In other embodiments, there is provided a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier, peptide, a cationselected from Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺, K⁺, Na⁺ or Al³⁺, or a mixturethereof, and a sterically hindered primary amine. In one embodiment, thecation is Mg²⁺, Ca²⁺ or Zn²⁺ or a mixture thereof. In a furtherembodiment, the pharmaceutical composition further comprises apharmaceutically acceptable binder and/or a pharmaceutically acceptableglidant, lubricant or additive that acts as both a glidant and lubricantand/or an antioxidant. In some embodiments, the pharmaceuticalcomposition is applied to a carrier. In some embodiments, the carrier isa filler.

In some cases the molar ratio of cation:sterically hindered primaryamine:peptide in the aqueous solution applied to the carrier is5-100:5-50:1. In some cases, the molar ratio of cation:stericallyhindered primary amine may be equal to or greater than 2:1 (e.g.,between 5:1 and 2:1). Thus, in some cases the molar ratio ofcation:sterically hindered primary amine:peptide applied to the carrieris 100:50:1, 100:30:1, 80:40:1, 80:30:1, 80:20:1, 60:30:1, 60:20:1,50:30:1, 50:20:1, 40:20:1, 20:20:1, 10:10:1, 10:5:1 or 5:10:1. Whenbinder, e.g., methylcellulose, is present in the GC-C agonist peptidesolution applied to the carrier it can be present at 0.5%-2.5% by weight(e.g., 0.7%-1.7% or 0.7%-1% or 1.5% or 0.7%).

In a further embodiment, the pharmaceutical composition furthercomprises a pharmaceutically acceptable binder or additive, and/or apharmaceutically acceptable glidant, lubricant or additive that acts asboth a glidant and lubricant and/or an antioxidant.

Suitable pharmaceutical compositions in accordance with the inventionwill generally include an amount of the active compound(s) with anacceptable pharmaceutical diluent or excipient, such as a sterileaqueous solution, to give a range of final concentrations, depending onthe intended use. The techniques of preparation are generally well knownin the art, as exemplified by Remington's Pharmaceutical Sciences (18thEdition, Mack Publishing Company, 1995).

The compositions described herein may be administered systemically orlocally, e.g.: orally (e.g. using capsules, powders, solutions,suspensions, tablets, sublingual tablets and the like), by inhalation(e.g. with an aerosol, gas, inhaler, nebulizer or the like), to the ear(e.g. using ear drops), topically (e.g. using creams, gels, liniments,lotions, ointments, pastes, transdermal patches, etc), ophthalmically(e.g. with eye drops, ophthalmic gels, ophthalmic ointments), rectally(e.g. using enemas or suppositories), nasally, buccally, vaginally (e.g.using douches, intrauterine devices, vaginal suppositories, vaginalrings or tablets, etc), via an implanted reservoir or the like, orparenterally depending on the severity and type of the disease beingtreated. The term “parenteral” as used herein includes, but is notlimited to, subcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intralesionaland intracranial injection or infusion techniques. Preferably, thecompositions are administered orally, intraperitoneally orintravenously.

For treatment of gastrointestinal disorders, the peptides describedherein are preferably administered orally, e.g., as a tablet, capsule,sachet containing a predetermined amount of the active ingredientpellet, gel, paste, syrup, bolus, electuary, slurry, powder, lyophilizedpowder, granules, as a solution or a suspension in an aqueous liquid ora non-aqueous liquid; as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion, via a liposomal formulation (see, e.g., EP736299) or in some other form. Orally administered compositions caninclude binders, lubricants, inert diluents, lubricating, surface activeor dispersing agents, flavoring agents, and humectants. Orallyadministered formulations such as tablets may optionally be coated orscored and may be formulated so as to provide sustained, delayed orcontrolled release of the active ingredient therein.

The peptides can be co-administered with other agents used to treatgastrointestinal disorders including but not limited to the agentsdescribed herein.

In another aspect, suitable pharmaceutical compositions may comprise oneor more other therapeutic agents. Such therapeutic agents include,without limitation, analgesic agents; anti-secretory agents, includingproton pump inhibitors, acid pump antagonists, H2 receptor antagonists;PDE5 inhibitors; GABA-B antagonists; bile acid sequestrants; prokineticand promotility agents; antidepressants; antibiotics; antiemetics; andmucosal-protecting agents.

Methods of Treatment

In various embodiments, the peptides and compositions described hereinare useful for the treatment of patient gastrointestinal disorder.

In some embodiments, the gastrointestinal disorder is selected from thegroup consisting of irritable bowel syndrome (IBS), constipation, afunctional gastrointestinal disorder, gastroesophageal reflux disease,functional heartburn, dyspepsia, visceral pain, gastroparesis, chronicintestinal pseudo-obstruction, colonic pseudo-obstruction, Crohn'sdisease, ulcerative colitis, and inflammatory bowel disease.

In a further embodiment, the gastrointestinal disorder is constipation.The constipation can be chronic constipation, idiopathic constipation,due to post-operative ileus, or caused by opiate use. Clinicallyaccepted criteria that define constipation include the frequency ofbowel movements, the consistency of feces and the ease of bowelmovement. One common definition of constipation is less than three bowelmovements per week. Other definitions include abnormally hard stools ordefecation that requires excessive straining (Schiller 2001, AlimentPharmacol Ther 15:749-763). Constipation may be idiopathic (functionalconstipation or slow transit constipation) or secondary to other causesincluding neurologic, metabolic or endocrine disorders. These disordersinclude diabetes mellitus, hypothyroidism, hyperthyroidism,hypocalcaemia, Multiple Sclerosis, Parkinson's disease, spinal cordlesions, Neurofibromatosis, autonomic neuropathy, Chagas disease,Hirschsprung's disease and Cystic fibrosis. Constipation may also be theresult of surgery (postoperative ileus) or due to the use of drugs suchas analgesics (like opioids), antihypertensives, anticonvulsants,antidepressants, antispasmodics and antipsychotics.

In other embodiments, the gastrointestinal disorder is irritable bowelsyndrome (IBS). The irritable bowel syndrome can beconstipation-predominant irritable bowel syndrome (c-IBS),diarrhea-predominant irritable bowel syndrome (d-IBS) or alternatingbetween the two irritable bowel syndromes (a-IBS).

In other embodiments, the gastrointestinal disorder is dyspepsia.

In other embodiments, the gastrointestinal disorder is gastroparesis.The gastroparesis can be selected from idiopathic, diabetic orpost-surgical gastroparesis.

In still other embodiments, the gastrointestinal disorder is chronicintestinal pseudo obstruction.

In other embodiments, the gastrointestinal disorder is Crohn's disease.

In some embodiments, the gastrointestinal disorder is ulcerativecolitis.

In some embodiments, the gastrointestinal disorder is inflammatory boweldisease.

In another embodiment, the gastrointestinal disorder is visceral pain.In a further embodiment, the present invention features a method fordecreasing gastrointestinal pain or visceral pain in a patient, themethod comprising, administering to the patient a pharmaceuticalcomposition comprising of peptide described herein. The peptide agonistsdescribed herein can be used alone or in combination therapy for thetreatment, prevention or reduction of visceral pain associated with agastrointestinal disorder or pain associated with another disorder.

In another embodiment, the invention features a method for treatinginflammation, including inflammation of the gastrointestinal tract,e.g., inflammation associated with a gastrointestinal disorder orinfection or some other disorder, the method comprising administering toa patient a pharmaceutical composition comprising a purified peptidedescribed herein.

In another embodiment, the invention features a method for treating agastrointestinal disorder comprising administering an agonist of theintestinal guanylate cyclase (GC-C) receptor either orally, by rectalsuppository, or parenterally.

In still another embodiment, the invention features a method fortreating a gastrointestinal disorder comprising administering an agonistof the intestinal guanylate cyclase (GC-C) receptor.

In another aspect, the invention features a method of increasing thelevel of cyclic guanosine 3′-monophosphate (cGMP) in a biologicalsample, tissue (e.g, the intestinal mucosa), or cell (e.g., a cellbearing GC-A receptor), or whole organism by contacting the sample,tissue, or organism to a peptides described herein.

The peptide GC-C receptor agonists described herein can be administeredin combination with other agents. For example, the peptides can beadministered with an analgesic peptide or compound. The analgesicpeptide or compound can be covalently attached to a peptide describedherein or it can be a separate agent that is administered together withor sequentially with a peptide described herein in a combinationtherapy. The peptides described herein may also be administered incombination with other agents used to treat GI disorders includingantidepressants, promotility or prokinetic agents, antiemetics,antibiotics, proton pump inhibitors, acid blockers (e.g., histamine H2receptor antagonists), acid pump antagonists, PDE5 inhibitors, GABA-Bagonists, bile acid sequestrants, and mucosal protecting agents.

In some embodiments, useful analgesic agents that may be used with thepeptides described herein include Ca channel blockers (e.g.,ziconotide), 5HT receptor antagonists (e.g., 5HT3, 5HT4 and 5HT1receptor antagonists), 5HT4 agonists (e.g., tegaserod [Zelnorm®],mosapride, zacopride, cisapride, renzapride, prucalopride [Resolor®],benzimidazolone derivatives such as BIMU 1 and BIMU 8, and lirexapride),5HT1 agonists (e.g., sumatriptan and buspirone), opioid receptoragonists (e.g., loperamide, fedotozine, enkephalin pentapeptide,morphine, diphenyloxylate, frakefamide, trimebutine and fentanyl), CCKreceptor agonists (e.g., loxiglumide and dexloxiglumide), NK1 receptorantagonists (e.g., aprepitant, vofopitant, ezlopitant, R-673(Hoffmann-La Roche Ltd), SR-48968 and SR-14033, (Sanofi Synthelabo),CP-122,721 (Pfizer, Inc.), GW679769 (Glaxo Smith Kline) and TAK-637(Takeda/Abbot)), NK2 receptor antagonists (e.g., nepadutant, saredutant,GW597599 (Glaxo Smith Kline), SR-144190 (Sanofi-Synthelabo) andUK-290795 (Pfizer Inc)), NK3 receptor antagonists (e.g., osanetant(SR-142801; Sanofi-Synthelabo), SR-241586 and talnetant),norepinephrine-serotonin reuptake inhibitors (NSRI) (e.g., milnacipran),mixed and selective dopamine receptor antagonists (e.g.—metoclopramide,itopride, domperidone), vanilloid and cannabanoid receptor agonists,sialorphin and sialorphin-related peptides. Analgesics agents in thevarious classes are described in the literature.

In some embodiments, one or more other therapeutic agents may be used incombination with the peptides described herein. Such agents includeantidepressants, promotility or prokinetic agents, antiemetics,antibiotics, proton pump inhibitors, acid blockers (e.g., histamine H2receptor antagonists), acid pump antagonists, PDE5 inhibitors, GABA-Bagonists, bile acid sequestrants, and mucosal protecting agents.

Examples of antidepressants include, without limitation, tricyclicantidepressants such as amitriptyline desipramine (Norpramin®),imipramine (Tofranil®), amoxapine (Asendin®), nortriptyline; theselective serotonin reuptake inhibitors (SSRI's) such as paroxetine(Paxil®), fluoxetine (Prozac®), sertraline (Zoloft®), and citralopram(Celexa®); and others such as doxepin (Sinequan®) and trazodone(Desyrel®).

Examples of promotility and prokinetic agents include, withoutlimitation, itopride, octreotide, bethanechol, metoclopramide (Reglan®),domperidone (Motilium®), erythromycin (and derivatives thereof) andcisapride (Propulsid®). An example of antiemetics includes, withoutlimitation, prochlorperazine.

Examples of antibiotics that may be used include those that may be usedto treat Helicobacter pylori infections, such as amoxicillin,tetracycline, metronidazole, or clarithromycin. Other antibiotics suchas erythromycin and derivatives thereof may also be used in combinationwith the peptides described herein.

Examples of proton pump inhibitors include, without limitation,omeprazole (Prilosec®), esomeprazole (Nexium®), lansoprazole(Prevacid®), pantoprazole (Protonix®) and rabeprazole (Aciphex®).Examples of H2 receptor blockers include, without limitation, includingcimetidine, ranitidine, famotidine and nizatidine. Examples of acid pumpantagonists include, without limitation, revaprazan, CS-526 (J.Pharmacol. Exp. Ther. (2007) 323:308-317), PF-03716556 (J. Pharmacol.Exp. Ther. (2009) 328(2):671-9), and YH1885 (Drug Metab. Dispos. (2001)29(1):54-9).

Examples of PDE5 inhibitors include, without limitation, avanafil,lodenafil, mirodenafil, sildenafil citrate, tadalafil, vardenafil andudenafil. GABA-B agonists include, without limitation, baclofen andXP19986 (CAS Registry No. 847353-30-4). Examples of bile acidsequestrants include, without limitation, GT102-279, cholestyramine,colesevelam, colesevelam hydrochloride, ursodeoxycholic acid,colestipol, colestilan, sevelamer, polydiallylamine cross-linked withepichlorohydrin, dialkylaminoalkyl derivatives of a cross-linkeddextran, and N-(cycloalkyl)alkylamines. Examples of mucosal protectingagents include, without limitation, sucralfate (Carafate), teprenone,polaprezinc, cetraxate and bismuth subsalicyclate.

Combination therapy can be achieved by administering two or more agents,e.g., a peptide described herein and another therapeutic peptide orcompound, each of which is formulated and administered separately, or byadministering two or more agents in a single formulation. Othercombinations are also encompassed by combination therapy. For example,two agents can be formulated together and administered in conjunctionwith a separate formulation containing a third agent. While the two ormore agents in the combination therapy can be administeredsimultaneously, they need not be. For example, administration of a firstagent (or combination of agents) can precede administration of a secondagent (or combination of agents) by minutes, hours, days, or weeks.Thus, the two or more agents can be administered within minutes of eachother or within 1, 2, 3, 6, 9, 12, 15, 18, or 24 hours of each other orwithin 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 days of each other orwithin 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks of each other. In some caseseven longer intervals are possible. While in many cases it is desirablethat the two or more agents used in a combination therapy be present inwithin the patient's body at the same time, this need not be so.

Dosage

The dose range for adult humans may be generally from 5 μg to 100 mg/dayorally of the peptides described herein. Tablets, capsules, or otherforms of presentation provided in discrete units may convenientlycontain an amount of compound described herein which is effective atsuch dosage or as a multiple of the same, for instance, units containing25 μg to 2 mg or around 100 μg to 1 mg. The precise amount of compoundprescribed to a patient will be the responsibility of the attendantphysician. However, the dose employed will depend on a number offactors, including the age and sex of the patient, the precise disorderbeing treated, and its severity.

In various embodiments, the dosage unit is administered with food atanytime of the day, without food at anytime of the day, with food afteran overnight fast (e.g. with breakfast), at bedtime after a low fatsnack. In one particular embodiment, the dosage unit is administeredprior to or subsequent to food consumption (e.g., a meal). In a furtherembodiment, the dosage unit is administered approximately 15 minutes to1 hour prior to food consumption. In various embodiments, the dosageunit is administered once a day, twice a day, three times a day, fourtimes a day, five times a day or six times a day. In certain embodimentsthe dosage unit and daily dose are equivalent.

The precise amount of each of the two or more active ingredients in adosage unit will depend on the desired dosage of each component. Thus,it can be useful to create a dosage unit that will, when administeredaccording to a particular dosage schedule (e.g., a dosage schedulespecifying a certain number of units and a particular timing foradministration), deliver the same dosage of each component as would beadministered if the patient was being treated with only a singlecomponent. In other circumstances, it might be desirable to create adosage unit that will deliver a dosage of one or more components that isless than that which would be administered if the patient was beingtreated only with a single component. Finally, it might be desirable tocreate a dosage unit that will deliver a dosage of one or morecomponents that is greater than that which would be administered if thepatient was being treated only with a single component.

The pharmaceutical composition can include additional ingredientsincluding but not limited to the excipients described herein. In certainembodiments, one or more therapeutic agents of the dosage unit may existin an extended or control release formulation and additional therapeuticagents may not exist in extended release formulation. For example, apeptide or agonist described herein may exist in a controlled releaseformulation or extended release formulation in the same dosage unit withanother agent that may or may not be in either a controlled release orextended release formulation. Thus, in certain embodiments, it may bedesirable to provide for the immediate release of one or more of theagents described herein, and the controlled release of one or more otheragents.

The present invention has been described with reference to certainexemplary embodiments thereof. However, it will be readily apparent tothose skilled in the art that it is possible to embody the invention inspecific forms other than those of the exemplary embodiments describedabove. This may be done without departing from the spirit of theinvention. The exemplary embodiments are merely illustrative and shouldnot be considered restrictive in any way. The scope of the invention isdefined by the appended claims and their equivalents, rather than by thepreceding description.

EXAMPLES

The GC-C agonist peptides or pharmaceutically acceptable salts thereofas described herein were prepared by solid phase chemical synthesis andnatural folding (air oxidation) by American Peptide Company (Sunnyvale,CA). In some cases, the peptides were modified after synthesis asdescribed herein.

The Cys₁-IMD peptide was synthesized by mixing 4.6 g (3.0 mmol) oflinaclotide in 200 ml of EtOH. Formaldehyde at 37% (1.12 ml/5 eq) wasadded to this mixture. The reaction mixture was incubated in a waterbath (45° C.) for overnight. The following day the solvent was removedby rota-evaporation. The peptide was further purified throughreverse-phase chromatography.

The Glu₃-ethyl ester peptide was synthesized on a 20 mmolFmoc-Tyr(tBu)-Wang resin. Protecting groups used for amino acids are:t-Butyl group for Tyr and Thr, Trt group for Asn and Cys. The peptidechain was assembled on the resin by repetitive removal of the Fmocprotecting group and coupling of protected amino acid. DIC and HOBt wereused as coupling reagents and NMM was used as the base for thisreaction. 20% piperidine in DMF was used as de-Fmoc-reagent. Afterremoval of last Fmoc protecting group, resin was treated with cocktail Kfor 3 hours to cleave the peptide from resin and removal of the sidechain protecting groups. The eluted peptide was precipitated in coldether and dried. The dried peptide was dissolved in a mixture ofTFA/TIS/water (95:3:2 v/v) in a ratio of 1 to 10 (g/v). This mixture wasstirred at room temperature for 1 hour. The isolated peptide was alsoprecipitated in cold ether, collected by filtration and dried under highvacuum.

The Tyr₁₄-ethyl ester peptide was synthesized by a fragment condensationmethod. Fragment A(Boc-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-OH)was prepared on 15 mmol CTC resin using Fmoc chemistry. This peptidechain was also assembled on the resin by repetitive removal of the Fmocprotecting group and coupling of protected amino acid. DIC and HOBt wereused as coupling reagents and NMM was used as the base. 20% piperidinein DMF was used as de-Fmoc-reagent. After removal of last Fmocprotecting group, Boc was coupled to protect the N-terminal amino group.The peptide resin was washed, dried, and treated with 1% TFA/DCM tocleave peptide from resin. Fragment B (Cys(Trt)-Tyr-OEt) was preparedfrom coupling of Fmoc-Cys(Trt)-OH and Tyr-OEt. HCl. The Fmoc group wasremoved by treating this di-peptide with 20% piperidine in DMF.

The Tyr₁₄-ethyl ester peptide was finally synthesized by coupling thetwo fragments in DMF. HBTU/HOBt/NMM was used as the coupling reagent forthis reaction. The protecting groups were removed by treating thepeptide with cocktail K for 2 hours. This peptide was precipitated incold ether and dried. The dried peptide was dissolved in a mixture ofTFA/TIS/water (95:3:2 v/v) in a ratio of 1 to 10 (g/v). This mixture wasstirred at room temperature for 1 hour. The isolated peptide was againprecipitated in cold ether, collected by filtration and dried under highvacuum.

Example 1: cGMP Accumulation in T84 Cells for Analysis of GC-C Activity

For the cGMP assay, 4.5×10⁵ cells/mL of T84 cells were grown overnightin 24 well tissue culture plates. On next day, the T84 cells were washedtwice with 1 mL of DMEM (pH 7). After the second wash, the cells wereincubated with 450 μL of 1 mM isobutylmethylxanthine (IBMX) in pH 7buffer for 10 minutes at 37° C. to inhibit any phosphodiesteraseactivity. The peptides were then diluted in DMEM buffer (pH 7) to a 10×concentration. The peptide solution of 50 μL was diluted to a finalvolume of 500 μL with the T84 cells, bringing each peptide concentrationto 1×. The peptides were tested in duplicate at 100 nM.

There was no peptide control used to determine endogenous levels ofcGMP. Peptides were incubated for 30 minutes at 37° C. After 30 minutes,the supernatants were removed and the cells were lysed with 0.1 M HCl.The cells were lysed for 30 minutes on ice. After 30 minutes, lysateswere pipetted off and placed into a 96 well HPLC plate and spun at10,000×G for 10 minutes to remove any cell debris. Supernatants from theprevious spin were removed and placed into a fresh 96 well HPLC plate.

cGMP concentrations were determined from each sample using the LC/MSconditions (Table 1 below) and calculated standard curve. EC₅₀ valueswere calculated from concentration-response curves generated withGraphPad Prism Software.

TABLE 1 LC/MS conditions MS: Thermo Quantum Ion Mode: ESI⁺ Scan Type:MRM Dwell Collision Retention Time Energy Tube Time Compound: Transition(msec) (V) Lens (min) cGMP 346 > 152 100 28 139 1.0 HPLC: AgilentTechnologies 1200 Series with CTC Analytics HTS PAL Column: ThermoHypersil Gold 2.1 × 50 mm 5 micron particle size Flow Rate: 400 uL/minColumn RT Temperature: Autosampler 6 ° C. Temperature: Injection Volume:20 uL Mobile Phases: A = 98:2 Water:Acetonitrile + 0.1% Formic Acid B =2:98 Water:Acetonitrile + 0.1% Formic Acid Gradient: Time (min) % A % B0 100 0 0.3 30 70 2.00 30 70 2.01 100 0 4 100 0

Example 2: Relative Binding Affinity of Exemplary Peptides to the GC-CReceptor of T84 Cells

The relative binding affinities of linaclotide and Cys₁-IMD to theguanylate cyclase-C receptor (GC-C) were determined using acompetitive-binding assay in which the peptides competed with a knownGC-C agonist, porcine-derived heat-stable enterotoxin (pSTa), forbinding sites on cell-surface GC-C receptors on human colonic epithelial(T84) cells. The pSTa was radiolabeled with ¹²⁵I to enable measurementof its receptor binding. The competitive-binding assay was performed byadding various concentrations of each peptide (0.1 to 3,000 nM) to 0.20mL reaction mixtures containing Dulbecco's modified Eagle's medium(DMEM), 0.5% bovine serum albumin (BSA), 2.0×10⁵ T84 cells, and 170 pM[¹²⁵I]-pSTa (200,000 cpm). The data were used to construct competitiveradioligand-binding curves and determine the relative binding affinitiesof linaclotide and Cys₁-IMD, as measured by their IC₅₀ and K_(i) values.Both linaclotide and Cys₁-IMD competitively inhibited the specificbinding of [¹²⁵I]-pSTa to cell-surface GC-C receptors on T84 cells.Their relative binding affinities, as measured by their inhibitionconstants (K_(i)), were as follows: Linaclotide K_(i)=3.9±1.6 nM andCys₁-IMD K_(i)=1.4±0.5 nM (FIG. 3 ).

Example 3: cGMP Response in T84 Cells Induced by Exemplary Peptides

Linaclotide and Cys₁-IMD were tested for guanylate cyclase-C(GC-C)agonist activity in T84 cells as follows. In each well of a 96-wellplate, approximately 200,000 T84 cells/well was first incubated with 1mM 3-isobutyl-1-methylxanthine (IBMX) in 0.18 mL of Dulbecco's modifiedEagle's medium (DMEM) for 10 minutes at 37° C. Each peptide was dilutedto final concentrations ranging from 0.1 to 10,000 nM, and 0.02 mL ofeach dilution was added in duplicate to a 96-well plate containing theT84 cells, for a final volume of 0.20 mL per well. The peptide reactionswere incubated for 30 min at 37° C. Following the incubation, thesupernatants were removed and discarded and the cells were lysed withcold 0.1 M hydrochloric acid (HCl) for 30 min on ice. The cell debriswas removed by centrifugation and the concentration of guanosine3′,5′-cyclic monophosphate (cyclic GMP) in each lysate was determinedusing liquid chromatography with tandem mass spectrometry. The data wereused to construct dose-response curves and calculate half-maximaleffective concentration (EC₅₀) values for each test article.

Linaclotide and Cys₁-IMD showed GC-C agonist activity in T84 cells, asmeasured by the increase in intracellular cGMP (FIG. 4 ). The EC₅₀values for linaclotide and Cys₁-IMD were 315±105 nM and 172±32 nM,respectively.

Example 4: Measurement of Content and Purity of Exemplary Peptides

Content and purity of the peptides of the present invention may bedetermined by reverse phase gradient liquid chromatography using anAgilent Series 1100 LC System with Chemstation Rev A.09.03 software orequivalent. A YMC Pro™ C18 column (dimensions: 3.0×150 mm, 3.5 μm, 120Å; Waters Corp., Milford, MA) or equivalent is used and is maintained at40° C. Mobile phase A (MPA) consists of water with 0.1% trifluoroaceticacid while mobile phase B (MPB) consists of 95% acetonitrile:5% waterwith 0.1% trifluoroacetic acid. Elution of the peptides is accomplishedwith a gradient from 0% to 47% MPB in 28 minutes followed by a ramp to100% MPB in 4 minutes with a 5 minute hold at 100% MPB to wash thecolumn. Re-equilibration of the column is performed by returning to 0%MPB in 1 minute followed by a 10 minute hold at 100% MPA. The flow rateis 0.6 mL/min and detection is accomplished by UV at 220 nm.

Samples for analysis are prepared by addition of the contents ofcapsules of exemplary peptides to 0.1 N HCl to obtain a targetconcentration of 20 μg peptide/mL. 100 μL of this solution is injectedonto the column.

Cys₁-IMD Peptide

The Cys₁-IMD peptide was purified using a 2-inch Waters C18 column with0.1% TFA buffer with a linear gradient of 15-45% in 60 minutes of bufferB at flow rate of 100 mL/min. The pooled fractions with purity around95% were loaded onto C18 column. After equilibrating the column withTEAP buffer and AA buffer, the peptide was purified and eluted out withHAC buffer with a linear gradient of 15-75% of buffer B in 60 minutes.Pooled fractions with purified peptide were lyophilized to dryness. Anexample of an analysis of linaclotide and Cys₁-IMD product by RP-HPLC isshown in FIG. 2 .

Glu₃-Ethyl Ester Peptide

The Glu₃-ethyl ester peptide (6.0 g) was dissolved in 12 L of 0.05Mammonium bicarbonate in water, and the oxidation process was monitoredby Ellman's test, MS and analytical HPLC. The oxidation process tookapproximately 48 hours for completion.

The above solution was filtered and loaded onto a 2-inch C18 column, andpurified by using 0.05M ammonium acetate buffer with a linear gradientof 10-40% of buffer B in 60 minutes at flow rate of 100 mL/min.

The pooled fractions with purity of >95% were lyophilized to dryness.After the peptides were dried, the peptide was re-dissolved inacetonitrile-water and acidified to pH around 4-5 by addition of aceticacid and re-lyophilized to dryness.

Tyr₁₄-Ethyl Ester Peptide

The Tyr₁₄-ethyl ester peptide was purified by dissolving 2.5 g of theisolated peptide in 5 L of 0.05M ammonium bicarbonate in water, and theoxidation process was monitored by Ellman's test, MS and analyticalHPLC. This oxidation process took approximately 16 hours for completion.

The above solution was filtered and loaded onto a 2-inch Polymer column,and purified by using 0.05M ammonium bicarbonate buffer with a lineargradient of 15-45% of buffer B in 60 minutes at flow rate of 100 mL/min.The pooled fractions with the peptide were lyophilized to dryness. Afterthe peptide was dried, the peptide was re-dissolved inacetonitrile-water and acidified to pH 4-5 by addition of acetic acidand re-lyophilized to dryness.

The contents of the purified peptides were measured by determining thepeptide concentration in the prepared sample against a similarlyprepared external peptide standard.

Other Embodiments

All publications and patents referred to in this disclosure areincorporated herein by reference to the same extent as if eachindividual publication or patent application were specifically andindividually indicated to be incorporated by reference. Should themeaning of the terms in any of the patents or publications incorporatedby reference conflict with the meaning of the terms used in thisdisclosure, the meaning of the terms in this disclosure are intended tobe controlling. Furthermore, the foregoing discussion discloses anddescribes merely exemplary embodiments of the present invention. Oneskilled in the art will readily recognize from such discussion and fromthe accompanying drawings and claims, that various changes,modifications and variations can be made therein without departing fromthe spirit and scope of the invention as defined in the followingclaims.

What is claimed is:
 1. A peptide or a pharmaceutically acceptable saltthereof, wherein the peptide comprises the amino acid sequence Cys CysGlu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr, wherein at least onecarboxyl group of the peptide is an alkyl ester having the formula(—COOR) in which R is a C₁₋₆ alkyl.
 2. The peptide or a pharmaceuticallyacceptable salt thereof according to claim 1, wherein the peptidecomprises the amino acid structure of:

or a pharmaceutically acceptable salt thereof, wherein R′ is H or a C₁₋₆alkyl, and at least one R′ is C₁₋₆ alkyl.
 3. The peptide orpharmaceutically acceptable salt thereof according to claim 1, whereinthe peptide comprises the amino acid structure of:

wherein R is a C₁₋₆ alkyl.
 4. The peptide or pharmaceutically acceptablesalt thereof according to any one of claims 1 or 3, wherein R is a C₁₋₄alkyl.
 5. The peptide or pharmaceutically acceptable salt thereofaccording to any one of claims 1, 3, or 4, wherein R is methyl.
 6. Thepeptide or pharmaceutically acceptable salt thereof according to any oneof claims 1, 3, or 4, wherein R is ethyl.
 7. The peptide orpharmaceutically acceptable salt thereof according to any one of claims1, 3, or 4, wherein R is propyl.
 8. The peptide or pharmaceuticallyacceptable salt thereof according to any one of claims 1, 3, 4, or 6wherein the peptide comprises the amino acid structure of:


9. The peptide or pharmaceutically acceptable salt thereof according toany one of claims 1, 3, 4, or 7 wherein the peptide comprises the aminoacid structure of:


10. The peptide or pharmaceutically acceptable salt thereof according toany one of claims 1, 3, 4, or 5 wherein the peptide comprises the aminoacid structure of:


11. The peptide or pharmaceutically acceptable salt thereof according toclaim 1, wherein the peptide comprises the amino acid structure of:

wherein R is C₁₋₆ alkyl.
 12. The peptide or pharmaceutically acceptablesalt thereof according to any one of claims 1 or 11, wherein R is a C₁₋₄alkyl.
 13. The peptide or pharmaceutically acceptable salt thereofaccording to any one of claims 1, 11, or 12, wherein R is methyl. 14.The peptide or pharmaceutically acceptable salt thereof according to anyone of claims 1, 11, or 12, wherein R is ethyl.
 15. The peptide orpharmaceutically acceptable salt thereof according to any one of claims1, 11, or 12, wherein R is propyl.
 16. The peptide or pharmaceuticallyacceptable salt thereof according to any one of claims 1, 11, 12, or 14,wherein the peptide comprises the amino acid structure of:


17. The peptide or pharmaceutically acceptable salt thereof according toany one of claims 1, 11, 12, or 15, wherein the peptide comprises theamino acid structure of:


18. The peptide or pharmaceutically acceptable salt thereof according toany one of claims 1, 11, 12, or 13, wherein the peptide comprises theamino acid structure of:


19. A peptide or a pharmaceutically acceptable salt thereof, wherein thepeptide comprises the amino acid structure of:


20. A peptide or a pharmaceutically acceptable salt thereof, wherein thepeptide consists of the amino acid sequence Cys Cys Glu Tyr Cys Cys AsnPro Ala Cys Thr Gly Cys Tyr, wherein at least one carboxyl group of thepeptide is an alkyl ester having the formula (—COOR) in which R is aC₁₋₆ alkyl.
 21. The peptide or a pharmaceutically acceptable saltthereof according to claim 20, wherein the peptide consists of the aminoacid structure of:

or a pharmaceutically acceptable salt thereof, wherein R′ is H or a C₁₋₆alkyl, and at least one R′ is C₁₋₆ alkyl.
 22. The peptide orpharmaceutically acceptable salt thereof according to claim 20, whereinthe peptide consists of the amino acid structure of:

wherein R is a C₁₋₆ alkyl.
 23. The peptide or pharmaceuticallyacceptable salt thereof according to any one of claims 20 or 22, whereinR is a C₁₋₄ alkyl.
 24. The peptide or pharmaceutically acceptable saltthereof according to any one of claims 20, 22, or 23, wherein R ismethyl.
 25. The peptide or pharmaceutically acceptable salt thereofaccording to any one of claims 20, 22, or 23, wherein R is ethyl. 26.The peptide or pharmaceutically acceptable salt thereof according to anyone of claims 20, 22, or 23, wherein R is propyl.
 27. The peptide orpharmaceutically acceptable salt thereof according to any one of claims20, 22, 23, or 25, wherein the peptide consists of the amino acidstructure of:


28. The peptide or pharmaceutically acceptable salt thereof according toany one of claims 20, 22, 23, or 26, wherein the peptide consists of theamino acid structure of:


29. The peptide or pharmaceutically acceptable salt thereof according toany one of claims 20, 22, 23, or 24, wherein the peptide consists of theamino acid structure of:


30. A peptide or pharmaceutically acceptable salt thereof according toclaim 20, wherein the peptide consists of the amino acid structure of:

wherein R is C₁₋₆ alkyl.
 31. The peptide or pharmaceutically acceptablesalt thereof according to any one of claims 20 or 30, wherein R is aC₁₋₄ alkyl.
 32. The peptide or pharmaceutically acceptable salt thereofaccording to any one of claims 20, 30, or 31, wherein R is methyl. 33.The peptide or pharmaceutically acceptable salt thereof according to anyone of claims 20, 30, or 31, wherein R is ethyl.
 34. The peptide orpharmaceutically acceptable salt thereof according to any one of claims20, 30, or 31, wherein R is propyl.
 35. The peptide or pharmaceuticallyacceptable salt thereof according to any one of claims 20, 30, 31, or33, wherein the peptide consists of the amino acid structure of:


36. The peptide or pharmaceutically acceptable salt thereof according toany one of claims 20, 30, 31, or 34, wherein the peptide consists of theamino acid structure of:


37. The peptide or pharmaceutically acceptable salt thereof according toany one of claims 20, 30, 31, or 32, wherein the peptide consists of theamino acid structure of:


38. A peptide or pharmaceutically acceptable salt thereof, wherein thepeptide consists of the amino acid structure of:


39. The peptide or pharmaceutically acceptable salt thereof according toany one of claims 1-38, wherein the peptide activates the guanylatecyclase C receptor.
 40. The peptide or pharmaceutically acceptable saltthereof according to any one of claims 1-19 or 39, wherein the peptidecomprises 30 or fewer amino acids.
 41. The peptide or pharmaceuticallyacceptable salt thereof according to any one of claims 1-19 or 39,wherein the peptide comprises 20 or fewer amino acids.
 42. The peptideor pharmaceutically acceptable salt thereof according to any one ofclaims 1-19 or 39, wherein fewer than five amino acids precede the firstCys residue of the amino acid sequence.
 43. The peptide orpharmaceutically acceptable salt thereof according to any one of claims1-42, wherein said peptide or pharmaceutically acceptable salt thereofis isolated.
 44. The peptide or pharmaceutically acceptable salt thereofaccording to any one of claims 1-43, wherein said peptide orpharmaceutically acceptable salt thereof is purified.
 45. Apharmaceutical composition comprising a peptide or pharmaceuticallyacceptable salt thereof according to any one of claims 1-44.
 46. Apharmaceutical composition comprising two or more peptides orpharmaceutically acceptable salts thereof according to any one of claims1-44.
 47. A pharmaceutical composition comprising two or more peptidesselected from: i. a peptide or a pharmaceutically acceptable saltthereof, wherein the peptide comprises the amino acid structure of:

ii. a peptide or a pharmaceutically acceptable salt thereof, wherein thepeptide comprises an amino acid structure of:

 and iii. a peptide or a pharmaceutically acceptable salt thereof,wherein the peptide comprises the amino acid sequence Cys Cys Glu TyrCys Cys Asn Pro Ala Cys Thr Gly Cys Tyr, wherein at least one carboxylgroup of the peptide is an alkyl ester having the formula (—COOR) inwhich R is a C₁₋₆ alkyl.
 48. A pharmaceutical composition comprisinglinaclotide and a peptide or a pharmaceutically acceptable salt thereof,wherein the peptide comprises an amino acid structure of:

and the peptide or pharmaceutically acceptable salt thereof comprisesless than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weightcompared to the weight of linaclotide.
 49. A pharmaceutical compositioncomprising linaclotide and a peptide or a pharmaceutically acceptablesalt thereof, wherein the peptide comprises an amino acid structure of:

and the peptide or pharmaceutically acceptable salt thereof comprisesless than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% by weight compared tothe weight of linaclotide.
 50. A pharmaceutical composition comprisinglinaclotide and a peptide or a pharmaceutically acceptable salt thereof,wherein the peptide comprises an amino acid structure of:

and the peptide or pharmaceutically acceptable salt thereof comprisesless than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weightcompared to the weight of linaclotide.
 51. A pharmaceutical compositioncomprising linaclotide and a peptide or a pharmaceutically acceptablesalt thereof, wherein the peptide comprises an amino acid structure of:

and the peptide or pharmaceutically acceptable salt thereof comprisesless than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% by weight compared tothe weight of linaclotide.
 52. A pharmaceutical composition comprisinglinaclotide and a peptide or a pharmaceutically acceptable salt thereof,wherein the peptide comprises an amino acid structure of:

and the peptide or pharmaceutically acceptable salt thereof comprisesless than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weightcompared to the weight of linaclotide.
 53. A pharmaceutical compositioncomprising linaclotide and a peptide or a pharmaceutically acceptablesalt thereof, wherein the peptide comprises an amino acid structure of:

and the peptide or pharmaceutically acceptable salt thereof comprisesless than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% by weight compared tothe weight of linaclotide.
 54. A pharmaceutical composition comprising apeptide or pharmaceutically acceptable salt thereof, wherein the peptideconsists of the amino acid structure of:

and the peptide comprises at least 90% by weight compared to the weightof linaclotide or another guanylate cyclase C agonist.
 55. Apharmaceutical composition consisting essentially of a peptide orpharmaceutically acceptable salt thereof, wherein the peptide comprisesthe amino acid structure of:


56. The pharmaceutical composition according to any one of claims 45-55,further comprising one or more agents selected from (i) a cationselected from Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺, K⁺, Na⁺ or Al³⁺, or (ii) asterically hindered primary amine.
 57. A pharmaceutical compositioncomprising a pharmaceutically acceptable carrier, a peptide according toany one of claims 1-44 and one or more agents selected from (i) a cationselected from Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺, K⁺, Na⁺ or Al³⁺, or (ii) asterically hindered primary amine.
 58. The pharmaceutical compositionaccording to claims 56 or 57, wherein said agent is Mg²⁺, Ca²⁺, Zn²⁺,Mn²⁺, K⁺, Na⁺ or Al³⁺.
 59. The pharmaceutical composition according toclaim 58, wherein said Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺, K⁺, Na⁺ or Al³⁺ isprovided as magnesium acetate, magnesium chloride, magnesium phosphate,magnesium sulfate, calcium acetate, calcium chloride, calcium phosphate,calcium sulfate, zinc acetate, zinc chloride, zinc phosphate, zincsulfate, manganese acetate, manganese chloride, manganese phosphate,manganese sulfate, potassium acetate, potassium chloride, potassiumphosphate, potassium sulfate, sodium acetate, sodium chloride, sodiumphosphate, sodium sulfate, aluminum acetate, aluminum chloride, aluminumphosphate or aluminum sulfate.
 60. The pharmaceutical compositionaccording to claims 56 or 57, wherein said agent is a stericallyhindered primary amine.
 61. The pharmaceutical composition according toclaim 60, wherein the sterically hindered primary amine is an aminoacid.
 62. The pharmaceutical composition according to claim 61, whereinthe amino acid is a naturally-occurring amino acid, a non-naturallyoccurring amino acid or an amino acid derivative.
 63. The pharmaceuticalcomposition according to claim 62, wherein the naturally-occurring aminoacid is histidine, phenylalanine, alanine, glutamic acid, aspartic acid,glutamine, leucine, methionine, asparagine, tyrosine, threonine,isoleucine, tryptophan or valine or the non-naturally occurring aminoacid is 1-aminocyclohexane carboxylic acid, lanthanine or theanine. 64.The pharmaceutical composition according to claim 60, wherein thesterically hindered primary amine has the formula:

wherein R₁, R₂ and R₃ are independently selected from: H, C(O)OH, C1-C6alkyl, C1-C6 alkyl ether, C1-C6 alkylthioether, C1-C6 alkyl carboxylicacid, C1-C6 alkyl carboxylamide and alkylaryl, wherein any group can besingly or multiply substituted with: halogen or amino, and provided thatno more than one of R₁, R₂ and R₃ is H.
 65. The pharmaceuticalcomposition according to claim 64, wherein the sterically hinderedprimary amine is cyclohexylamine or 2-methylbutylamine.
 66. Thepharmaceutical composition according to claim 60, wherein the stericallyhindered primary amine is a polymeric amine.
 67. The pharmaceuticalcomposition according to claim 66, wherein the polymeric amine ischitosan.
 68. The pharmaceutical composition according to any one ofclaims 60-67, wherein said pharmaceutical composition further comprisesMg²⁺, Ca²⁺, Zn²⁺, Mn²⁺, K⁺, Na⁺ or Al³⁺.
 69. The pharmaceuticalcomposition according to claim 68, wherein said Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺,K⁺, Na⁺ or Al³⁺ is provided as magnesium acetate, magnesium chloride,magnesium phosphate, magnesium sulfate, calcium acetate, calciumchloride, calcium phosphate, calcium sulfate, zinc acetate, zincchloride, zinc phosphate, zinc sulfate, manganese acetate, manganesechloride, manganese phosphate, manganese sulfate, potassium acetate,potassium chloride, potassium phosphate, potassium sulfate, sodiumacetate, sodium chloride, sodium phosphate, sodium sulfate, aluminumacetate, aluminum chloride, aluminum phosphate or aluminum sulfate. 70.The pharmaceutical composition according to any one of claims 45-69,further comprising an antioxidant.
 71. The pharmaceutical compositionaccording to claim 70, wherein said antioxidant is BHA, vitamin E orpropyl gallate.
 72. The pharmaceutical composition according to any oneof claims 45-71, further comprising a pharmaceutically acceptable binderor additive.
 73. The pharmaceutical composition according to claim 72,wherein the pharmaceutically acceptable binder or additive is selectedfrom polyvinyl alcohol, polyvinylpyrrolidone (povidone), a starch,maltodextrin or a cellulose ether.
 74. The pharmaceutical compositionaccording to claim 73, wherein the pharmaceutically acceptable binder oradditive is polyvinyl alcohol.
 75. The pharmaceutical composition ofclaim 73, wherein the pharmaceutically acceptable binder or additive isa cellulose ether.
 76. The pharmaceutical composition of claim 75,wherein the cellulose ether is selected from: methylcellulose,ethylcellulose, carboxymethylcellulose, hydroxyethyl cellulose,hydroxyethyl methylcellulose, hydroxypropyl cellulose and hydroxypropylmethylcellulose.
 77. The pharmaceutical composition of any of claims45-76, further comprising a pharmaceutically acceptable filler.
 78. Thepharmaceutical composition according to claim 77, wherein thepharmaceutically acceptable filler is cellulose, isomalt, mannitol ordibasic calcium phosphate.
 79. The pharmaceutical composition of claim78, wherein the cellulose is selected from microfine cellulose andmicrocrystalline cellulose.
 80. The pharmaceutical composition accordingto any one of claims 45-79, further comprising an additional therapeuticagent.
 81. The pharmaceutical composition according to claim 80, whereinsaid additional therapeutic agent is selected from one or more of ananalgesic agent, an antidepressant, a promotility or prokinetic agent,an antiemetic, an antibiotic, a proton pump inhibitor, an acid blocker,a PDE5 inhibitor, an acid pump antagonist, a GABA-B agonist, a bile acidsequestrant or a mucosal protecting agent.
 82. A dosage unit comprisinga pharmaceutical composition according to any one of claims 45-81. 83.The dosage unit according to claim 82, wherein said dosage unit is acapsule or tablet.
 84. The dosage unit according to claim 83, whereineach of said dosage units comprises 5 μg to 1 mg of said peptide.
 85. Amethod for treating a gastrointestinal disorder comprising administeringthe pharmaceutical composition according to any one of claims 45-81. 86.The method of claim 85, wherein the gastrointestinal disorder isselected from the group consisting of: irritable bowel syndrome (IBS),constipation, a functional gastrointestinal disorder, gastroesophagealreflux disease, functional heartburn, dyspepsia, visceral pain,gastroparesis, chronic intestinal pseudo-obstruction, colonicpseudo-obstruction, Crohn's disease, ulcerative colitis, andinflammatory bowel disease.
 87. The method of claim 86, wherein thegastrointestinal disorder is constipation.
 88. The method of claim 87,wherein the constipation is chronic constipation, idiopathicconstipation, due to post-operative ileus, or caused by opiate use. 89.The method of claim 86, wherein the gastrointestinal disorder isirritable bowel syndrome (IBS).
 90. The method of claim 89, wherein theirritable bowel syndrome is constipation-predominant irritable bowelsyndrome (c-IBS), diarrhea-predominant irritable bowel syndrome (d-IBS)or alternating between the two irritable bowel syndromes (a-IBS). 91.The method of claim 86, wherein the gastrointestinal disorder isdyspepsia.
 92. The method of claim 86, wherein the gastrointestinaldisorder is gastroparesis.
 93. The method according to claim 92, whereinsaid gastroparesis is idiopathic, diabetic or post-surgicalgastroparesis.
 94. The method of claim 86, wherein the gastrointestinaldisorder is chronic intestinal pseudo obstruction.
 95. The method ofclaim 86, wherein the gastrointestinal disorder is Crohn's disease. 96.The method of claim 86, wherein the gastrointestinal disorder isulcerative colitis.
 97. The method of claim 86, wherein thegastrointestinal disorder is inflammatory bowel disease.
 98. The methodof claim 86, wherein the gastrointestinal disorder is visceral pain. 99.A method for increasing intestinal motility in a patient, the methodcomprising administering to the patient an effective amount of thepharmaceutical composition according to any one of claims 45-81.
 100. Amethod of increasing guanylate cyclase C (GC-C) receptor activity in abiological sample or organism, comprising contacting said biologicalsample or organism with a peptide according to any one of claims 1-44.